Phytoestrogenic isoflavone compositions, their preparation and use thereof for protection against and treatment of radiation injury

ABSTRACT

The present invention provides compositions and methods for the prophylactic and therapeutic treatment of animals, including humans from radiation injury. In particular, the present invention provides methods and compositions comprising the isoflavone genistein (4′,5,7-trihydroxyflavone) or phytoestrogenic isoflavonoids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/297,978, filed Jun. 10, 2004, which is the National Stage ofInternational Application No. PCT/US01/19089 filed Jun. 12, 2001 whichclaims benefit to U.S. Provisional Application No. 60/223,734 filed Aug.8, 2000 and U.S. Provisional Application No. 60/211,375 filed Jun. 14,2000. Each of these applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for theprophylactic and therapeutic treatment of animals, including humans,from radiation injury. In particular, the present invention providesmethods and compositions comprising the isoflavone genistein(4′,5,7-trihydroxyflavone) and other phytoestrogenic isoflavonoids.

BACKGROUND OF THE INVENTION

Radioprotective agents are compounds that reduce the biological effectsof radiation (for review, see e.g., Hall, Radiobiology for theRadiobiologist, Lippincott Williams & Wilkins, Philadelphia, Pa.[1994]). They may be administrate before and/or after radiation exposureand can protect the organism from radiation-induced lethality.Radioprotectors have been shown to operate by a variety of differentmechanisms (for review, see e.g., Bump and Malaker (eds.),Radioprotectors: Chemical, Biological, and Clinical Perspectives, CRCPress, Washington, D.C. [1997]). These include their antioxidantproperties (Weiss and Landauer, Ann. NY Acad. Sci., 899:4460 [2000]),their estrogenic activity (Miernicki et al., Soc. Neurosci. Abstr.,16:1054 [1990]; and Patt et al., Amer. J. Physiol., 159:269-280 [1949]),and/or in some cases, their ability to inhibit protein kinase(s)involved in signal transduction (Liu et al., Oncogene, 19: 571-579[2000]).

A variety of antioxidant compounds has been shown to confer radiationprotection. These range from the highly toxic aminothiols to theantioxidant vitamins. However, the majority of these compounds have sideeffects of varying severity. For example, sulfhydryl radioprotectorssuch as amifostine (See e.g., U.S. Pat. No. 5,994,409) are highly toxicto mammals and must be administered in the hospital setting. Adverseside-effects associated with these compounds include nausea andvomiting, hypotension, hypocalcemia and drowsiness (Bienvenu et al.,Adv. Exp. Med. Biol., 264:291-300 [1990]). Amifostine acts by scavengingfree radicals (Murray, in Bump and Malaker, supra). Antioxidant vitamins(A, C, E and beta carotene) provide only minimal levels of radiationprotection, have a very short window of protection, and if obtained fromdietary sources, must be eaten in a variety of foods, since any singlefood source only has small levels of any vitamin (Weiss and Landauer,supra).

In addition, using presently used methods and compositions, it isnecessary to administer single high doses of agents such aspharmaceuticals or other chemical additives by parenteral routes withina short time frame before or after the radiation or chemical insult (Seee.g., Bump and Malaker, supra). Therefore, this precludes their use as along-term prophylactic measure for use in protection againstunanticipated radiation injury. Because of the short duration of actionof most radioprotective agents, there has been a long and on-goingsearch for agents that confer long lasting protection. Indeed, thereremains a great need for a nontoxic, orally or parenterally availableradioprotective agent that can be made available both before and afterradiation injury.

DESCRIPTION OF FIGURES

FIG. 1 shows the structure of the isoflavonoid genistein (4′,5,7trihydroxyflavone).

FIG. 2 shows the structure of the phytoestrogen genistein showingsimilarities with the structure of the hormone estradiol.

FIG. 3 shows the histogram for the effect of a single oral (po)administration of genistein on 30-day survival. Mice were givengenistein either 1 hour or 24 hours before an 8.5 Gy dose of gammaradiation from a cobalt-60 source. The dose rate was 0.6-Gy/minute.Experimental groups consisted of saline, polyethylene glycol (PEG)vehicle, or genistein (400 mg/kg). While genistein did not offerprotection 1 hour before radiation, 88% of mice that received genistein24 hours survived, compared to 63% survival for the saline and PEGcontrol groups (N=16/group). The asterisk indicates a significantdifference from control.

FIG. 4 shows the survival curve of mice administered a single oral (po)administration of saline, polyethylene glycol (PEG) vehicle, or 400mg/kg genistein 1 hour before 8.5 Gy gamma radiation (N=16/group).Survival was monitored for 30 days postirradiation. This figure depictsthe survival curve for the data illustrated in FIG. 3.

FIG. 5 shows the 30 day survival curve for mice administered a singleoral (po) dose saline, polyethylene glycol (PEG) vehicle, or 400 mg/kggenistein 24 hours before 8.5 Gy gamma radiation (N=16, group). Survivalwas monitored for 30 days postirradiation. This figure depicts thesurvival curve for the data illustrated in FIG. 3.

FIG. 6 shows the histogram for the effect of a single oral (po)administration of genistein on 30-day survival of mice given genisteineither 1 hour or 24 hours before a 9.5 Gy dose of gamma radiation from acobalt-60 source. The dose rate was 0.6 Gy/minute. Experimental groupsconsisted of saline, polyethylene glycol (PEG) vehicle, or genistein(400 mg/kg) (N=24-32/group).

FIG. 7 shows the survival curve of mice administered a single oral (po)administration of saline, polyethylene glycol (PEG) vehicle, or 400mg/kg genistein 1 hour before 9.5 Gy gamma radiation (N=24-32/group).Survival was monitored for 30 days postirradiation. This figure depictsthe survival curve for the data illustrated in FIG. 6.

FIG. 8 shows the 30-day survival curve for mice administered a singleoral (po) dose of saline, polyethylene glycol (PEG) vehicle, or 400mg/kg genistein 24 hours before 9.5 Gy gamma radiation. Survival wasmonitored for 30 days postirradiation (N=24-32/group). This Figuredepicts the survival curve for the data illustrated in FIG. 6. Althoughgenistein did not protect against lethality, the data indicate thatgenistein treated mice survived for about a week longer than controlanimals.

FIG. 9 shows a histogram of the percent survival of irradiated micetreated with multiple daily oral (po) treatment with saline,polyethylene glycol (PEG) vehicle, or genistein (100 mg/kg or 400 mg/kg)(N=16/group). Mice were either treated with genistein for 4 days before9.5 Gy gamma radiation (pre), 4 days after 9.5 Gy radiation (pre), or 4days before and 4 days after 9.5 Gy radiation (pre+post). Survival wasmonitored for 30 days. The asterisk indicates a significant differencefrom control.

FIG. 10 shows the 30-day survival curve of mice treated with multipledaily oral (po) treatment with saline, polyethylene glycol (PEG)vehicle, or 100 mg/kg genistein (N=16/group). Mice were either treatedwith genistein for 4 days before 9.5 Gy gamma radiation (pre), 4 daysafter 9.5 Gy radiation (pre), or 4 days before and 4 days after 9.5 Gyradiation (pre+post). Survival was monitored for 30 days. This Figuredepicts the survival curve for the data illustrated in FIG. 9.

FIG. 11 shows the 30-day survival curve of mice treated with multipledaily oral (po) treatment with saline, polyethylene glycol (PEG)vehicle, or 400 mg/kg genistein. Mice were either treated with genisteinfor 4 days before 9.5 Gy gamma radiation (pre), 4 days after 9.5 Gyradiation (post), or 4 days before and 4 days after 9.5 Gy radiation(pre+post) (N=16/group). Survival was monitored for 30 days. This Figuredepicts the survival curve for the data illustrated in FIG. 9.

FIG. 12 shows the histogram for the effect of a single subcutaneous (sc)administration of genistein on 30-day survival of mice given genistein24 hours before a 9.5 Gy dose of gamma radiation form a cobalt-60source. The dose rate was 0.6 Gy/minute. Experimental groups consistedof saline, polyethylene glycol (PEG) vehicle, or genistein (100 mg/kg or400 mg/kg) (N=16/group). A single dose of genistein administered at 100or 400 mg/kg injected subcutaneously significantly protected mice from alethal dose of gamma radiation. The asterisk indicates a significantdifference from control.

FIG. 13 shows the thirty-day survival curve for mice receiving a singlesubcutaneous (sc) injection of either saline, polyethylene glycol (PEG)vehicle, 100 mg/kg genistein or 400 mg/kg genistein, 24 hours before a9.5 Gy dose of gamma radiation (N=16/group). This Figure depicts thesurvival curve for the data illustrated in FIG. 12.

FIG. 14 shows the effects of a single oral (po) administration ofsaline, polyethylene glycol (PEG) vehicle, or genistein on locomotorbehavior of mice over 48 hrs. Locomotor activity is expressed as thetotal distance traveled. The solid bars on the abscissa represent thedark period and the open bars the daylight period of the 12:12 hourlight/dark cycle. Each mouse received a single oral gavage (per os, po)of saline, PEG vehicle, or 50, 100, 200, or 400 mg/kg genisteinimmediately before testing at the beginning of the dark period on thefirst day (N=8/group). Vertical lines represent the SEM. There were nosignificant differences among groups on locomotor activity, indicatingthat orally Administered genistein is nontoxic using this sensitivebehavioral assay.

FIG. 15 shows the effects of a single subcutaneous (so) administrationof saline, polyethylene glycol (PEG) vehicle, or genistein on Locomotorbehavior of mice over 48 hrs. Locomotor activity is expressed as thetotal distance traveled. The solid bars on the abscissa represent thedark period and the open bars the daylight period of the 12:12 hourlight/dark cycle. Each mouse received a single subcutaneous dose ofsaline, PEG vehicle, or 50, 100, 200, or 40D mg/kg genistein immediatelybefore testing at the beginning of the dark period on the first day(N=8/group). Vertical lines represent the SEM. There were no significantdifferences among groups on locomotor activity indicating thatsubcutaneously administered genistein is nontoxic using this sensitivebehavioral assay.

FIG. 16 shows the effect of genistein on forelimb grip strength for miceevaluated on days 1, 4 and 14 after acute subcutaneous administration ofsaline, PEG vehicle, or 100, 200, or 400 mg/kg of genistein. Day 0 wasthe day of injection. As indicated, there were no significantdifferences among groups.

FIG. 17 shows the effect of genistein on forelimb grip strength for miceevaluated on days 1, 4 and 14 after acute oral gavage of saline, PEGvehicle, or 100, 200, or 400 mg/kg of genistein. Day 0 was the day ofinjection. There were no significant differences among groups.

FIG. 18 shows the effect of genistein on motor coordination as measuredby the inverted screen test for mice evaluated on days 1, 4, and 14after acute subcutaneous administration of saline, PEG vehicle, or 100,200, or 400 mg/kg of genistein. As indicated, there were no significantdifferences among groups.

FIG. 19 shows the effect of genistein on the motor coordination usingthe inverted screen test for mice evaluated on days 1, 4, and 14 afteracute oral administration of saline, PEG vehicle, or 100, 200, or 400mg/kg of genistein. As indicated, there were no significant differencesamong groups.

FIG. 20 shows the mean (SEM) body weight of mice administered an acutesubcutaneous dose of saline, PEG vehicle, or 100, 200, or 400 mg/kg ofgenistein. As indicated, there were no significant differences amonggroups.

FIG. 21 shows the mean SEM body weight of mice administered an acuteoral dose of saline, PEG vehicle, or 100, 200, or 400 mg/kg ofgenistein. As indicated, there were no significant differences amonggroups.

FIG. 22 shows the effect of a single oral gavage or subcutaneousinjection of saline, PEG vehicle, or 400 mg/kg of genistein on testesweights. Weights reflect the sum, 14 days after injection, of bothtestes with epididymes removed. Vertical lines represent the mean ″ SEM.As indicated, there were no significant differences among groups.

FIG. 23 provides a histogram showing the effect of a single subcutaneousdose of genistein on 30-day survival. Genistein (3.125-400 mg/kg) wasadministered 24 hr before 9.5 Gy radiation at a dose rate of 0.6/Gyminute.

FIG. 24 provides a 30-day survival curve for mice given a singlesubcutaneous injection of genistein. Mice received doses of 3.125-400mg/kg 24 hours before 9.5 Gy cobalt-60 irradiation. Significantly moremice survived 30 days after lethal dose of radiation if they hadreceived 25 to 400 mg/kg of genistein 24 hours before radiationexposure.

SUMMARY OF THC INVENTION

The present invention provides compositions and methods for theprophylactic and therapeutic treatment of animals, including humans,from radiation injury. In particular, the present invention providesmethods and compositions comprising the isoflavone genistein(4′,5,7-trihydroxyflavone) and other phytoestrogenic isoflavonoids.

The present invention provides methods for the radioprotection of asubject comprising providing a subject, a composition comprising atleast one isoflavonoid, and a radiation source, administering thecomposition to the subject, and exposing the subject to radiationproduced by a radiation source. In some preferred embodiments, thecomposition is administered to the subject before the subject is exposedto radiation, while in other preferred embodiments, the composition isadministered to the subject after the subject has been exposed toradiation. In particularly preferred embodiments, the subject isprotected from tissue damage from radiation. In some preferredembodiments, the subject is normal, while in other preferredembodiments, the subject is suffering from disease or anotherabnormality. In additional embodiments, the isoflavonoid(s) is selectedfrom the group consisting of genistin, genistein, 6″-O-Mal genistein,6″-O—Ac genistein, daidzein, 6″-O′Mal daidzein, 6″-O—Ac daidzein,glycitein, glycitin, 6″-O-Mal glycitin, biochannin A, formononetin, andmixtures thereof. In some embodiments, the isoflavonoid(s) is anantioxidant. In still further embodiments, the isoflavonoid(s) hasestrogenic activity, while in alternative embodiments, theisoflavonoid(s) is a tyrosine kinase inhibitor. In additionalembodiments, the isoflavonoid(s) comprises an angiogenesis inhibitor. Inyet other embodiments, isoflavonoid(s) lowers the low-densitylipoprotein concentration in the blood of the subject, and in otherembodiments, the isoflavonoid(s) comprises a vasodilatory agent. In somepreferred embodiments, the isoflavonoid is obtained from a sourceselected from the group consisting of soy, soy products and clover. Inparticularly preferred embodiments, the isoflavonoid is selected fromthe group consisting of genistin, genistein conjugates, genisteinderivatives, genistein analogues, natural genistein, and syntheticgenistein. In still other preferred embodiments, the isoflavonoid isdissolved in a vehicle. In some particularly preferred embodiments, thevehicle is polyethylene glycol. In additional embodiments, thecomposition further comprises one or more pharmaceutically acceptablecarriers, excipients, auxiliaries, and diluents.

In some embodiments of the methods, the composition is systemicallyadministered. Some preferred embodiments, the composition isadministered in a pharmaceutically acceptable form, while in otherpreferred embodiments, the composition is administered in the diet ofthe subject or as a dietary supplement administered to the subject. Insome embodiments, the composition is administered to the subject in asingle dose, while in other embodiments, the composition is administeredto, the subject in multiple doses. In preferred embodiments, theadministering is selected from the group consisting of subcutaneousinjection, oral administration, intravenous administration, rectaladministration, vaginal administration, topical administration,intramuscular administration, intranasal administration, transdermaladministration, subconjunctival administration, intraocularadministration, periocular administration, retrobulbar administration,subretinal, suprachoroidal administration, and intrathecaladministration. In alternative embodiments, the administering isadministration from a source selected from the group consisting ofmechanical reservoirs, devices, implants, and patches. In still furtherembodiments, the composition is in a form selected from the groupconsisting of pills, capsules, liquids, gels, powders, suppositories,suspensions, creams, jellies, aerosol sprays, and dietary supplements.In some preferred embodiments, the dietary supplement comprises anunprocessed soy food, while in other preferred embodiments, the dietarysupplement comprises isolated soy protein. In additional embodiments,the isoflavonoid is a natural ingredient of a dietary component.

In some embodiments, the composition comprises from about 0.1 mg toabout 2000 mg isoflavonoid. In some preferred embodiments, the dosage ofthe composition administered to the subject is from about 5 mg/day toabout 2000 mg/day isoflavonoid, while in other preferred embodiments,the dosage of the composition administered to the subject comprises fromabout 25 mg/day to about 1200 mg/day isoflavonoid, or from about 40mg/day to about 1200 mg/day isoflavonoid, or in yet further embodiments,the dosage of the composition administered to the subject comprises fromabout 30 mg/day to about 200 mg/day isoflavonoid. In other embodiments,the composition is administered to the subject is in a dosage of aneffective amount less than about 400 mg/kg/day of the body weight of thesubject. In some preferred embodiments, the composition is administeredto the subject is in a dosage of an effective amount from about 1mg/kg/day to 20 mg/kg/day of the body weight of the subject.

In some embodiments, the composition is administered from about 10minutes to 96 hours before radiation exposure. In some additionalembodiments, the composition is administered as a single dose, while inother embodiments the composition is administered in multiple doses ofthe same or varying concentration of isoflavonoid(s). In otherembodiments, the composition is administered from about 1 minute to 48hours after radiation exposure. In some embodiments, the radiation isselected from the group consisting of ionizing radiation, alpharadiation, beta radiation, gamma radiation, neutrons, microwaves, andelectromagnetic radiation.

The present invention also provides nontoxic radiation protectivecompositions comprising a therapeutically effective amount of at leastone nontoxic, phytoestrogenic isoflavonoid selected from the groupconsisting of genistin, genistein, 6″-O-Mal genistein, 6″-O—Acgenistein, daidzein, 6″-O-Mal daidzein, 6″-O—Ac daidzein, glycitein,glycitin, 6″-O-Mal glycitin, biochanin A, formononetin, and mixturesthereof. In some preferred embodiments, the therapeutically effectiveamount is a prophylactically effective amount.

The present invention further provides methods for preparing nontoxic,radiation-protective compositions comprising at least one isoflavonoid,comprising the steps of dissolving an isoflavonoid selected from thegroup consisting of genistin, genistein, 6″-O-Mal genistein, 6″-O—Acgenistein, daidzein, 6″-O-Mal daidzein, 6″-O—Ac daidzein, glycitein,glycitin, 6″-O-Mal glycitin, biochanin A, formononetin, and a mixturethereof in a vehicle selected from the group consisting of polyethyleneglycol (PEG) and sesame oil vehicle to produce a suspension; andseparating the isoflavonoid of the suspension to produce an isoflavonoidsolution. In some embodiments, the composition further comprises atleast one additional ingredient selected from the group consisting ofpharmaceutically acceptable carriers, excipients, auxiliaries, anddiluents.

DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for theprophylactic and therapeutic treatment of animals, including humans,from radiation injury. In particular, the present invention providesmethods and compositions comprising the isoflavone genistein(4′,5,7-trihydroxyflavone) and other phytoestrogenic isoflavonoids.

Indeed, the present invention provides means to reduce the mortalityassociated with radiation exposure by animals, including humans.Accordingly, the present invention provides a method for prophylacticand therapeutic treatment of radiation damage to animals, includinghumans.

In particularly preferred embodiments, the present invention providesnontoxic, naturally occurring dietary supplements for the use asradiation protective agents. However, it is not intended that thepresent invention be limited to dietary supplements, as the presentinvention finds use through various other means of administration,including but not limited to subcutaneous, intramuscular, intravenous,etc.

Other compounds have been investigated for their radioprotectiveproperties. For example, estrogen has been found to reduceradiation-induced lethality (Miernicki et al., supra; Patt et al.,supra). However, Miernicki et al. reported that radioprotective doses ofthe estrogen 17-beta-estradiol were behaviorally toxic. As aphytoestrogen (Messina, Am. J. Clin. Nutr., 70 (3 Suppl): 439S-450S[1999]), it was believed that genistein may have enough estrogenicactivity to be protective, but not as to be toxic. A comparison of thesimilarities in structure of genistein and estradiol are illustrated inFIG. 2.

I. Genistein

As indicated herein, genistein (4′,5,7-trihydroxyflavone) (See, FIG. 1)is considered a potent antioxidant (Wei et al., Proc. Soc. Exp. Biol.Med., 208:124-30 [1995]). Genistein also inhibits DNA topoisomerase II,cell cycle progression, and angiogenesis. In addition, it has been shownto be a vasodilatory agent and to reduce LDL (low-density lipoprotein)cholesterol levels. Indeed, these properties were the bases of studiesinvestigating alternative mechanisms of genistein's protective action(Kim, Am. J. Clin. Nutr., 68:1418 S-1425S [1998]). Furthermore, ionizingradiation-induced apoptosis (programmed cell death) is triggered bytyrosine kinase activation. Genistein, as an inhibitor of proteintyrosine kinases, has been shown to prevent radiation-induced cell death(Uckun et al., Clin. Canc. Res., 4:1125-34 [1998]). Therefore, proteinkinase inhibitors such as genistein were investigated as candidateradioprotective agents during the development of the present invention.

However, in contrast to the present invention, in which genistein isutilized to prolong the survival of an animal following exposure toradiation, genistein has been investigated as an adjunct therapy forcancer treatment to enhance the killing and/or suppression of tumorcells. For example, genistein, in conjunction with X-rays, was found tocause an enhancement of radiation-induced cell death (van Rijn and vanden Berg, Clin. Canc. Res., 3:1775-9 [1997]). The soybean-derivedBowman-Birk Inhibitor (BBI), a protease inhibitor not related to theisoflavone genistein, suppresses x-ray induced transformation of cellsbut does not protect human lung cancer cells from radiation-inducedcytotoxicity. In fact, treatment with the BBI inhibitor actuallyenhanced cell killing by cisplatin in combination with radiationtreatment in the lung carcinoma cells (Kennedy et al., Nutr. Canc.,26:209-17 [1996]).

U.S. Pat. No. 5,824,702, discusses the use of genistein to protect theskin from ultraviolet radiation. However, this Patent is limited to thetopical application of genistein for protection against ultravioletradiation. Ultraviolet radiation, because it is less capable ofpenetrating through matter than is visible light, is considered for allpractical purposes to be non-ionizing (See e.g., Attix, Introduction toRadiological Physics and Radiation Dosimetry. John Wiley & Sons, NewYork [1986]). Moreover, the window of efficacy for this use of genisteinis indicated as being limited to two hours. Thus, the use of aprotectant that must be applied to the skin and has a short window ofefficacy against essentially non-ionizing radiation is quite differentfrom the protectant of the present invention which protects againsthighly penetrating ionizing radiation (e.g., gamma rays).

U.S. Pat. No. 6,071,956, discusses the use of flavonoids as inhibitorsof heat shock protein, the formation of which is an injurious biologicalconsequence of tissue stress. Radiation is cited among the applicablestressors, however, this Patent is limited to inhibitingradiation-induced heat shock proteins as they relate to smooth muscletissue injury.

II. Radioprotection

During the development of the present invention, genistein was found tohave the characteristics of an ideal radioprotectant. However, it iscontemplated that additional compounds will find use in the presentinvention (e.g., daidzein and glycitein and their metabolites).Advantages to the use of genistein include its nontoxic, antioxidant,phytoestrogenic, protein tyrosine kinase inhibitor properties. Inaddition, it is a natural product available in the diet from a singlefood source, and can be given daily to provide a long window ofprotective efficacy. Furthermore, it can be easily administered and hasan established long shelf life.

Nonetheless, prior to the development of the present invention, the useof genistein to provide effective radiation protection against and/orameliorating the potentially lethal effects of ionizing radiation wasapparently unknown, as no reference discusses the use of genistein toprotect animals from ionizing radiation injury or death. Although anunderstanding of the mechanism(s) is not necessary in order to use thepresent invention, it is believed that the combination of theantioxidative, estrogenic, and protein tyrosine-kinase inhibitoryproperties of genistein provides protection from ionizing radiationinjury or death.

Shimoi and colleagues (Shimoi et al., Carcinogenesis, 15:2669-72[1994]), reported that a single gastric intubation of a variety offlavonoids, including genistein, given six hours before irradiation,reduced the frequency of micronucleated reticulocytes in peripheralblood of mice. However, whole organism radioprotection was neverdemonstrated or mentioned. Indeed, Shimoi's work was limited toinvestigations of cells, rather than an entire animal.

Uma and colleagues (Uma et al., Radiat. Res., 151:74-8 [1999]) reportedthat two flavonoids, orientin and vicenin, isolated from the leaves of amedicinal plant, offered some protection (60-67% survival) whenintraperitoneally (IP) administered to mice 30 minutes before aradiation dose 1.3 Gy above the LD₅₀. The compounds were less effective(30-35% protection) when given orally, intravenously, orintramuscularly. Agents administered to mice by IP injection oftenconfer the best protection presumably because drugs can be absorbeddirectly from the peritoneal fluid. In humans, however, IP is not anormal route for drug administration. In addition, the compounds had alimited time window of efficacy before irradiation (30-60 minutes, ascompared with 1-4 day efficacy provided by the present invention) andwere not effective at all if given after radiation. Furthermore, theywere not evaluated under a multiple dose regimen or via the subcutaneousroute, which are common methods of delivery in humans. However, in thepresent invention, survival of multiple doses of oral genisteinadministration resulted in survival of 69% at a radiation dosecomparable to that of Uma et al., where only 30-35% survival wasobtained. Moreover, in the present invention, using the subcutaneousroute of administration 100 and 400 mg/kg doses of genistein providedsurvival rates of 81% and 88%, respectively. This is a substantialincrease in radioprotection over that reported for the non-isoflavoneflavonoids evaluated by Uma et al. In addition, the compounds Uma et al.evaluated have no documented estrogenic or protein tyrosine kinaseinhibitory activity. In contrast, although an understanding of themechanism(s) is not necessary in order to use the present invention, andit is not intended that the present invention be so limited, it iscontemplated that the superior efficacy of genistein is due to asynergistic effect of combined antioxidant, estrogenic, and proteintyrosine kinase inhibitory properties.

As indicated in the Examples, experiments conducted during thedevelopment of the present invention clearly show that the isoflavonegenistein is a very effective radiation protective agent. As discussed,it was shown to protect against radiation-induced lethality and enhancesurvival when administered one day before radiation exposure, by boththe oral and subcutaneous routes of administration. In addition, ifgiven in multiple oral doses, genistein enhances 30-day survival whengiven both 4 days before, as well as 4 days before and 4 days after alethal dose of gamma radiation, thus providing a long window ofprotective efficacy. Importantly, behavioral tests demonstrated that atall doses (50-400 mg/kg) evaluated, genistein did not result in anybehavioral toxicity. Thus, experiments conducted during the developmentof the present invention led to the development of novel combinations ofnontoxic, natural food sources, with effective radioprotection, and along window of protection.

The present invention finds wide use in various settings. Indeed, itfinds use anywhere where radiation is likely to be a problem. Forexample, the present invention finds use in protection against a solarradiation event, such as those potentially experienced by astronauts(Parsons and Townsend, Radial. Res., 153:729-33 [2000]), as well as bypilots and other flight personnel that make frequent high altitude tripswhere radiation exposure is a potential hazard (Bottollier-Depois etal., Radial. Res., 153:526-32 [2000]). In addition, the presentinvention finds use in conjunction with radiation therapy in the clinic,nuclear power plant facilities, food radiation plants, and in cleanup ofradiation dump sites and accidents (e.g., such as those experienced inChernobyl, Ukraine, Tokaimura, Japan, and Three-Mile Island, U.S.). Itis also contemplated that the present invention will find use by themilitary in the event of a nuclear radiation event, as well as bycivilian civil defense personnel in response to a terrorist radiationevent. It is further contemplated that the present invention will finduse in reducing the toxic effects of inhaled radionuclides and inreducing toxicity from radiation produced by electronic devices such ascellular phones.

It is further contemplated that because reactive oxygen species andrelated free radicals may be generated with equal effect as a result ofboth radiation and chemotherapy, antioxidant isoflavonoids such asgenistein will find use as effective agents in mitigating the toxiceffects of chemotherapy. “Chemoprotection” refers to protection fromchemicals such as chemotherapeutic agents exemplified by cisplatin andthe like.

The present invention provides ideal protective agents, as they arenontoxic, produce no behavioral alterations or other side effects, arenaturally occurring, have minimal cost, and a long shelf life. Inaddition, the compositions of the present invention are suitable fordaily use. Therefore, diet-derived products that offer the radiation andchemoprevention of the present invention are contemplated to findwidespread long-term use (Kelloff et al., J. Nut., 130(2SSuppl):467S-471S [2000]).

III. Preferred Embodiments

In some preferred embodiments, the present invention provides methodsthat comprise the administration of an isoflavone in an amountsufficient to treat radiation injury prophylactically ortherapeutically. Although it is contemplated that any isoflavone willfind use in the present invention, in preferred embodiments, genistein,a related isoflavonoid or metabolite with the same properties (e.g.,excellent antioxidant properties and estrogenic properties) are used. Inan alternative embodiments, the related isoflavonoid or metabolite hasprotein kinase activity.

The isoflavone protector, preferably genistein, can be administered byany suitable route. Genistein may be administered by mouth (per os), byinjection, in the diet, or by any number of other systemic routes. Theisoflavone protector can be administered either singly or in multipledosing regimens either before or before and after exposure to ionizingradiation. For the prophylactic treatment of radiation injury, theisoflavone is administered preferably orally or by subcutaneousinjection. If given orally, in particularly preferred embodiments, it isadministered from about 24 or more hours before the radiation exposureand repeated dosing of this compound, by appropriate dietary means orother means is preferred. However, it is not intended that the presentinvention be limited to this particular time frame, as in sonic cases,oral administration less than 24 hours prior to radiation exposure ispreferred. When administered subcutaneously, single administration 1-24hrs before exposure is suitable. However, it is not intended that thepresent invention be limited to this particular time frame, as in somecases, subcutaneous administration less or more than 24 hours prior toradiation exposure is preferred. Regardless of the route ofadministration, in some embodiments, increased beneficial effects areobserved if continued after the radiation event.

In addition to the active ingredients, the compositions may of thepresent invention contain suitable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used either in dietary supplements, foods,feeds, and/or as pharmaceutical preparations. Further details ontechniques for formulation and administration for pharmaceuticalpreparations may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

In addition to dietary administration (i.e., in food, feed, dietarysupplements, etc.), pharmaceutical compositions for oral administrationcan be formulated using pharmaceutically acceptable carriers well knownin the art in dosages suitable for oral administration. Such carriersenable the pharmaceutical compositions to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,and the like, for ingestion by the subject. Pharmaceutical preparationsfor oral use can be obtained through combination of active compoundswith solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude carbohydrate or protein fillers, such as sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gumsincluding arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid, or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers. Thus,any suitable vehicle finds use in the present invention.

Dietary and pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

Dietary and pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes).

After the dietary and pharmaceutical compositions have been prepared,they are placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of genistein, such labelingwould include amount, frequency, and method of administration.

Dietary and pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose is well within the capability ofthose skilled in the art.

For any compound, the prophylactically and/or therapeutically effectivedose can be estimated initially either in cell culture assays (e.g., ofneoplastic cells), or in animal models, usually mice Animal models mayalso be used to determine the appropriate concentration range and routeof administration. Such information is then used to determine usefuldoses and routes for administration in humans.

As indicated herein, prophylactically and therapeutically effectivedoses refer to that amount of active ingredient (e.g., genistein) whichameliorates the symptoms and/or effects of radiation. Efficacy andtoxicity may be determined by standard pharmaceutical procedures in cellcultures or experimental animals (e.g., ED₅₀, namely, the dose that istherapeutically effective in 50% of the population), and LD₅₀ (i.e., thedose that is lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the “therapeutic index,” and it can beexpressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration. One advantage of the present invention is lowtoxicity. Thus, the therapeutic index for compounds such as genistein isquite high.

The exact dosage used with each subject is typically determined eitherby the subject, medical profession (e.g., physician, nurse, etc.),and/or nutritionist/dietary counselor, taking into consideration factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, amount ofradiation exposure experienced or to be experienced, general health ofthe subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

Typically, dosage amounts vary from about 5 to 2000 mg/day, or at levelsup to approximately 16 mg/kg/day. In some embodiments, lowerconcentrations are preferred (e.g., 600-1200 mg/day). While in stillfurther embodiments, even lower concentrations are preferred (e.g.,about 200 mg/day). Indeed, the dosage amounts vary, depending upon thepatient, as indicated above. Guidance as to particular dosages andmethods of delivery is provided in the literature and generallyavailable to practitioners in the art.

It is contemplated that the present invention will benefit the worldcommunity. Indeed, the present invention provides the additionalprotection against the potential threats of insults from the innocent orintentional exposure of the population to the harmful effects ofradiation. The source of this exposure can range from the rapid growthin the use of radiation emitting household appliances, medical devicesand high powered electrical transmission systems, including wirelesscommunication systems or devices (i.e., cell phones) to the threat ofnuclear disasters.

DEFINITIONS

As used herein, the term “animal” refers to any animal, includinghumans. The term “non-human animal” includes vertebrates andinvertebrate animals, including but not limited to rodents, arthropods,insects, fish, non-human primates, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc. Inpreferred embodiments, the term refers to mammals, while in particularlypreferred embodiments, the term refers to humans.

As used herein, the term “subject” refers to any animal, including, butnot limited to humans. However, in preferred embodiments, the animal isa mammal (e.g., humans, rodents, non-human primates, ovines, bovines,ruminants, lagomorphs, porcines, caprines, equines, canines, felines,etc.). In some embodiments, the subject is “normal,” while in otherembodiments, the subject is suffering from pathology (e.g., infectiousdisease, cancer, genetic or inherited diseases, etc.). In particularlypreferred embodiments, the subject is treated using the method(s) andcomposition(s) of the present invention.

As used herein, the term “treatment” refers to the administration of theradioprotective composition in an amount that is effective in preventinginjury to an animal who has been or will be exposed to radiation. Thus,in some embodiments, the term encompasses the administration of theradioprotective composition prior to the exposure of the animal toradiation (i.e., “prophylactic” administration), while in otherembodiments the term encompasses treating an animal after the animal hasbeen exposed to the radiation (i.e., “therapeutic” administration). Instill further embodiments, the term encompasses the continuation of theamelioration of the injury long after the radiation exposure.

As used herein, the term “antioxidant” refers to compounds that have theability to slow the oxidation rate of oxidizable substances, inparticular those that are autoxidizable. Antioxidants act throughseveral chemical and physiological means, including chelation with metalions, scavenging of free radicals, and termination of chain reactionsthat occur during lipid peroxidation.

As used herein, the term “phytoestrogen” refers to weak estrogeniccompounds produced by plants.

As used herein, the term “flavonoid” refers to a group of phenoliccompounds found in fruits and vegetables. The basic structure offlavonoids consists of two benzene rings linked through a heterocyclicpyran ring (See e.g., Kuo, Organogenesis 8:47-69 [1997]).

As used herein, the term “isoflavonoid” refers to a subclass offlavonoids characterized by the presence of a second benzene attached tothe C3 position of C2. These compounds include genistein, daidzein,glycitein, as well as their glucosides and metabolites. In addition, thesubclass includes 4-methyl ether derivatives of genistein and daidzein,biochanin A, and formonectin, as well as genistin, 6″-O-Mal genistein,6″-O—Ac genistein, 6″-O-Mal daidzein, 6″-O—Ac daidzein, glycitin, and6″-O-Mal glycitin.

As used herein, the term “radiation” refers to any form ofelectromagnetic radiation. Absorbed doses are typically measured in“grays” (Gy).

As used herein, the term “ionizing radiation” refers to radiation thathas sufficient energy to eject one or more orbital electrons from anatom or molecule (e.g., α particles, β particles, γ rays, x-rays,neutrons, protons, and other particles having sufficient energy toproduce ion pairs in matter.

As used herein, the term “vehicle” refers to any composition that issuitable for use as a diluent, solvent, or other composition suitablefor producing a suspension of a compound of interest. In preferredembodiments, the vehicle is a liquid, colloidal, or semi-solidcomposition, such as water, polyethylene glycol, oil (e.g., sesame oil),or other liquid suitable for producing a suspension comprising at leastone flavonoid or isoflavonoid. In some embodiments, the vehicle of thepresent invention is a solid that contains at least one isoflavonoid orflavonoid. Thus, the term also encompasses dietary sources (includingdietary supplements) of genistein and/or other flavonoids.

The term “mixture” refers to a mingling together of two or moresubstances without the occurrence of a reaction by which they would losetheir individual properties. The term “solution” refers to a liquidmixture. The term “aqueous solution” refers to a solution that containssome water. In many instances, water serves as the diluent for solidsubstances to create a solution containing those substances. In otherinstances, solid substances are merely carried in the aqueous solution(i.e., they are not dissolved therein). The term aqueous solution alsorefers to the combination of one or more other liquid substances withwater to form a multi-component solution.

The term “parenterally” refers to administration to a subject throughsome means other than through the gastrointestinal tract. The mostcommon mode of parenteral administration is intravenous. However, othermodes of parenteral administration include, but are not limited to,intramuscular, intradermal, intrathecal, intranasal and subcutaneousadministration.

As used herein, the term “pharmaceutical composition” refers tocompositions composed of one or more pharmaceutically acceptablediluents, excipients or carriers. As used herein, the phrase“pharmaceutical preparation suitable for parenteral administration”refers to a solution containing at least one flavonoid and/orisoflavonoid compound in a pharmaceutically acceptable form forparenteral administration. The characteristics of the form will dependon a number of factors, including the mode of administration: Forexample, a preparation for intravenous administration will oftencomprise the compound dissolved in normal saline or sterile water forinjection. Of course, the pharmaceutical preparations of the presentinvention are not limited to those diluents; indeed, other components ordiluents known in the field of pharmaceuticals and pharmacy are withinthe scope of the present invention. The pharmaceutical preparation maycontain diluents, adjuvants and excipients, among other components,provided that those additional components neither adversely effect thepreparation (e.g., they do not cause degradation of the compound) northe recipient (e.g., they do not cause a hypersensitivity reaction).

As used herein, the term “topically active agent” indicates a substanceor composition which elicits a pharmacologic response at the site ofapplication. In preferred embodiments, the agent is a radioprotectivecomposition, while in particularly preferred embodiments, the agent isgenistein.

As used herein, the term “systemically active agent” is used broadly toindicate a substance or composition which will produce a pharmacologicresponse at a site remote from the point of application. In preferredembodiments, the agent is a radioprotective composition, while inparticularly preferred embodiments, the agent is genistein.

As used herein, the term “medical devices” includes any material ordevice that is used on, in, or through a patient's body in the course ofmedical treatment for radiation therapy. Medical devices include, butare not limited to, such items as medical implants, drug deliverydevices, and body cavity and personal protection devices. The medicalimplants include, but are not limited to, urinary catheters,intravascular catheters, dialysis shunts, skin sutures, vascular grafts,implantable meshes, intraocular devices, heart valves, and the like.

As used herein, the term “dietary supplement” refers to as product thatcontains one or more of the following dietary ingredients: a vitamin, amineral, an herb or other botanical, an amino acid, a dietary substancefor use by humans and other animals to supplement the diet by increasingthe total dietary intake, and/or a concentrate, metabolite, constituent,extract of any of these ingredients. Thus, it is intended that the termencompass any dietary supplement that comprises a flavonoid, inparticular isoflavonids such as genistein. It is not intended that thepresent invention be limited to any particular dietary supplement(s), asthe flavonoid of the present invention finds use as a dietary supplementadministered alone or in combination with other dietary supplements.

As used herein, the terms “food” and “feed” refer to food suitable forhuman and/or non-human animal use. The terms encompass liquid, solid,semi-solid, and other nutritional substances.

As used herein, the term “diet” refers to the nutritional intake of asubject (e.g., an animal). It is intended that the term encompass food,feed, dietary supplements, and other items ingested by a subject to meetnutritional, energy, and other bodily requirements.

The term “substantially purified,” as used herein, refers to a compoundthat is removed from its natural environment, isolated or separated, andare at least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which it is naturally associated. In someembodiments, the term refers to compounds synthesized in the laboratoryin which the compound is at least 60% free, preferably 75% free, andmost preferably 90% free from other components with which it isassociated during the synthetic process.

As used herein, the term “purified” refers to the removal ofcontaminants from a sample. Methods such as carbon, hydrogen andnitrogen analyses (CHN analysis, or “elemental analysis”) may be used todetermine the purity of compounds. In preferred embodiments, the CHNvalues of compounds of the present invention are very close to thepredicted values. Correspondence of experimental with the predictedvalues to within 0.3% indicates high levels of purity.

EXPERIMENTAL

The following Examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply; eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); Gy(gray or grays; 1 Gy=100 rad); po (per os; by mouth); sc (subcutaneous);iv (intravenous); ip (intraperitoneal); im (intramuscular); ° C.(degrees Centigrade); Sigma (Sigma Chemical Co., St. Louis, Mo.);Charles River (Charles River, Raleigh, N.C.); Jackson (JacksonLaboratory, Bar Harbor, Me.); Boehringer Mannheim (Boehringer Mannheim,Indianapolis, Ind.); Fisher (Fisher Scientific, Pittsburgh, Pa.); LifeTechnologies (Life Technologies, Rockville, Md.); Abbott (AbbottLaboratories, North Chicago, Ill.); Ultrasonics (Ultrasonics, Plainview,N.Y.); Omnitech (Omnitech Electronics, Columbus, Ohio); Chatillon(Chatillon, Greensboro, N.C.).

The examples presented herein are intended to be illustrative in natureand in no way intended to limit the scope of this invention.

In the experiments described herein, male CD2F1 mice (Charles River)weighing 24-30 grams were used. All mice were quarantined on arrival andrepresentative animals were screened for evidence of disease. Mice werehoused in a facility accredited by the Association for Assessment andAccreditation of Laboratory Animal Care International. Animal rooms weremaintained at 21+/−2° C., with 50%+/−10% humidity on a 12/12 hrlight/dark cycle. Commercial rodent ration (Harlan Teklad Rodent Diet8604) was freely available as was acidified (pH, 2.5-2.8) water tocontrol opportunistic infections (See, McPherson, Lab. Animal Care,13:737-44 [1963]). Mice were housed in polycarbonate cages in groups ofeight.

In the experiments described herein, compounds: Control groups receivedeither saline (Abbott) or the drug vehicle, polyethylene glycol with amolecular weight of 400 (PEG). PEG vehicle and genistein were obtainedfrom Sigma. PEG is a viscous, slightly hygroscopic liquid, which findswide use in food and food packaging as well as in the pharmaceuticalindustry. During the development of the present invention, severalstudies were conducting involving drugs dissolved in PEG-400 because ofits high solubility and low radioprotective characteristics. However,there appeared to be no reports in which genistein has been solubilizedin PEG-400. After many attempts to solubilize genistein, it wasdetermined that after brief sonication (10 seconds, medium pulse) usinga sonicator cell disrupter (Model W255R, Heat Systems Ultrasonics), highconcentrations of genistein could be easily solubilized in PEG-400.Thus, 100 mg genistein was solubilized in 1 ml of PEG-400 for theseexperiments. The solution obtained was injected sc at 0.1 ml/mouse ordiluted to 0.25 ml/mouse for oral administration. This concentrationcorresponds to 400 mg/kg body weight for a 25 gram animal.

In Examples 1-3 below, mice were irradiated in the bilateralgamma-radiation field of the Armed Forces Radiobiology ResearchInstitute cobalt-60 facility (Carter and Verrelli, AFRRI cobaltwhole-body irradiation (Technical Report 73-3); Bethesda, Md.: ArmedForces Radiobiology Research Institute [1973]). The midline tissue (MLT)dose to the animals was 8.5-9.5 Gy. The dose rate was 0.6 Gy/min. Thedose rate was established in an acrylic mouse phantom by use of a0.5-cc, tissue-equivalent ionization chamber (calibration factortraceable to the National Institute of Standards and Technology). Thedose conversion factor (DCF) 0.96 and the field were uniform to within+/−3%. Dose measurements followed the American Association of Physicistsin Medicine protocol (American Association of Physicists in Medicine,Med. Phys., 10:741-771 [1983]). MLT doses were determined by applyingthe DCF to dose measurements made free in air (FIA). The DCF wasdetermined by taking the ratio of two measurements. The firstmeasurement was the MLT dose-rate taken at a well-defined point in theabdominal region of the phantom. The second measurement was the FIAtissue dose-rate, taken after removing the phantom, at a convenientpoint in the region that the phantom had occupied. Once determined for aparticular experimental setup, the DCF value can be applied to allfuture FIA measurements to obtain the MLT dose-rate using the samesetup.

Example 1 Protective Effects of a Single Oral Dose

This Example describes experiments to determine the protective effectsof a single oral dose of genistein administered 1 or 24 hours beforeradiation mitigates radiation-induced mortality. The effects ofgenistein on radioprotection was evaluated after a single oral (po) doseof saline, PEG vehicle, or 400 mg/kg genistein (genistein-400)administered 1 hour or 24 hours before 8.5 Gy or 9.5 Gy cobalt-60 gammaradiation delivered at a dose rate of 0.6 Gy/min (N=16). Followingirradiation, mice were returned to their home cages where survival wasmonitored for 30 days. The radiation. LD50/30 for male CD2F1 miceadministered saline was determined to be approximately 8.3 Gy. The30-day survival rate was analyzed using a Chi square test.

As shown in FIGS. 3 and 4, the results demonstrated genistein did notenhance survival when given 1 hour before 8.5 Gy radiation. However,when given 24 hours before 8.5 Gy radiation, 63% of saline and vehiclecontrol mice survived 30 days after irradiation while 88% of micereceiving a single dose of genistein survived, as shown in FIGS. 3 and5. These results indicate that genistein has radioprotective qualitiesat this dose of radiation. When a higher does of radiation (9.5 Gy) wasadministered genistein given 1 hour or 24 hours before irradiation didnot enhance survival, as shown in FIGS. 6 through 8. However, the micethat received 400 mg/kg genistein 24 hours before radiation lived forabout a week longer than control animals indicating a beneficial effectof genistein (See, FIG. 8). To determine if multiple oral doses ofgenistein given daily before or before and after radiation would enhancesurvival, another experiment was conducted, as described in Example 2,below.

Example 2 Protective Effect of Multiple Oral Doses of Genistein

This Example describes experiments to determine the protective effect ofmultiple oral doses of genistein administered before or before and afterradiation. In these experiments, mice (N=16/group) received po 100 mg/kgor 400 mg/kg genistein for either 4 days before (pre), 4 days after(post), or 4 days before and 4 days after (pre+post) a lethal dose ofgamma radiation (9.5 Gy) Animals in the pre-irradiation genistein groupsreceived the PEG vehicle after irradiation, and the postirradiationgenistein groups received the PEG vehicle before irradiation. Thus, allof the animals received eight daily oral gavage of either vehicle orgenistein. The postirradiation dosing began 1 hour after irradiation.Two control groups that received either saline or PEG both before andafter irradiation were also included. This resulted in a total of eighttreatment conditions: 1) saline control; 2) PEG vehicle control; 3)genistein-100 pre; 4) genistein-100 post; 5) genistein-100 pre+post; 6)genistein-400 pre; 7) genistein-400 post; and 8) genistein-400 pre+post.

The results indicated that multiple dosing of genistein are capable ofprotecting animals from radiation-induced lethality at relatively highdoses (9.5 Gy) of gamma radiation. FIG. 9 provides the 30=day survivalrates for saline, PEG vehicle, were only 0%, and 19%, respectively. Asindicated in FIGS. 9 and 10, the survival rates for genistein-100 pre,post, and pre+post were 0%, 0% and 50%, respectively, while the survivalrates for the genistein-400 pre, post, and pre+post groups were 44%, 0%,and 69%, respectively, as indicated in FIGS. 9 and 11. These experimentsdemonstrate that multiple oral doses of the isoflavone genistein arecapable of protecting animals against a lethal dose of radiation.

Example 3 Subcutaneous Administration of Genistein

In this Example, experiments using another route of administration,namely a single subcutaneous injection of genistein administered 24hours before radiation. In these experiments, mice received a singlesubcutaneous (so) injection in the nape of the neck with either saline,PEG vehicle, genistein 100 mg/kg, or genistein 400 mg/kg, 24 hoursbefore a lethal dose of radiation. Both these doses of genistein weredemonstrated to be nontoxic using the sensitive locomotor activity test,as described herein.

Of the control mice that received saline or PEG vehicle, only 13% and6%, respectively survived 30 days after total body radiation exposure.In contrast, significantly more mice survived in the groups thatreceived a single dose of genistein, as indicated in FIGS. 12 and 13.Indeed, 69% of mice in the genistein-100 group survived, while 81% ofthose mice receiving genistein-400 survived. These results clearlydemonstrate that a single dose of genistein administered subcutaneouslyis a very potent radioprotective agent.

Example 4 Behavioral Toxicity

In this Example, experiments conducted to determine the behavioraltoxicity of a single oral dose of genistein that has been determined tobe radioprotective are described. Behavioral experiments were conductedin non-irradiated mice to determine the effects of genistein onlocomotor activity, a sensitive index of behavioral toxicity that iswell-known in the art (MacPhail, J. Am. Coll. Toxicol., 8:117-125[1989]).

In these experiments, computerized digiscan activity monitors (Omnitech)were used to quantify locomotor activity as previously described(Landauer et al., J. Radiat. Res., 38:45-54 [1997]). Each monitor usedan array of infrared photodetectors spaced 2.5 cm apart to determine thetotal distance traveled. Immediately after an oral gavage (po) ofsaline, PEG vehicle, or 50, 100, 200 or 400 mg/kg genistein, the mice(N=8/group) were each placed into an individual Plexiglas activitychamber (20 cm×20 cm×30 cm). Locomotor activity testing commenced at thebeginning of the dark cycle and continued for 48 hrs. Each animal wastested only once. Food and water were available throughout the testingperiod. No animals used in the behavioral studies were irradiated. Ananalysis of variance was used to statistically analyze locomotoractivity data.

The behavioral studies revealed that all doses of genistein (50-400mg/kg) administered orally by gavage (po) or subcutaneously byinjection, had no effect on locomotor activity, as shown in FIG. 14. Thebehavior of the genistein treated animals were no different than thecontrol groups, indicating that genistein has no motor side effects asindicated by this sensitive behavioral test.

Example 5 Behavioral Toxicity of Subcutaneous Genistein Injection

In this Example, experiments to determine whether a single subcutaneousinjection of genistein has any behavioral side effects, as measured bythe locomotor activity test of Example 4 are described.

Immediately after receiving a subcutaneous (so) administration ofsaline, PEG vehicle, or 50, 100, 200 or 400 mg/kg genistein, the mice(N=8/group) were each placed into locomotor activity monitors asdescribed above for 48 hours. The results indicate that scadministration of genistein had no effect on locomotor activityindicating that genistein did not result in behavioral toxicity, asshown in FIG. 15.

Example 6 Behavioral Toxicity Measured by the Grip Strength and MotorCoordination Tests

In this Example, experiments conducted to determine whether a singleoral or subcutaneous injection of genistein produces behavioral toxicityas measured by the grip strength test or a motor coordination test aredescribed. As discussed further below, these results further demonstratethe absence of toxicity at doses of 100, 200, or 400 mg/kg administeredacutely either orally or subcutaneously.

I. Behavioral Experiments

Behavioral experiments were conducted using 10 groups of non-irradiatedmice (N=10/group) to determine the acute effects of genistein onforelimb grip strength and motor coordination, using the inverted screentest. Each mouse, by group, received a single subcutaneous injection orsingle gavage of saline, PEG, or 100, 200, or 400 mg/kg of genistein.All drugs were administered subcutaneously with an injection volume of0.1 ml. The day of injection was considered to be “day 0.”

II. Grip-Strength Test

Forelimb grip strength performance was assessed using an establishedprocedure (Meyer et al., Neurobehav. Toxicol; 1:233-236 [1979]). Peakforelimb grip strength was measured in kilograms by a Chatillon DigitalForce Gauge (Model DFI2). The gauge was attached to a stainless steelT-bar. A mouse was placed with its forepaws on the T-bar and gentlypulled backward by the tail at a steady rate until its grip was broken.In order to eliminate bias, the individual administering the gripstrength test was unaware of the treatment received by the animal. Twotrials per mouse were conducted and the average of these trials wascomputed to estimate forelimb grip strength. The tests were conducted ondays 1, 4, and 14 after genistein administration during the lightportion of the light/dark cycle.

FIG. 16 shows the effect of genistein on forelimb grip strength for miceevaluated on days 1, 4 and 14 after acute subcutaneous, administrationof saline, PEG vehicle, or 100, 200, or 400 mg/kg of genistein. Day 0was the day of injection. As indicated, there were no significantdifferences among groups.

FIG. 17 shows the effect of genistein on forelimb grip strength for miceevaluated on days 1, 4 and 14 after acute oral gavage of saline, PEGvehicle, or 100, 200, or 400 mg/kg of genistein. Day 0 was the day ofinjection. There were no significant differences among groups.

III. Motor Coordination Assessment

In addition to the grip strength test, motor coordination was assessedusing the inverted screen test (Coughenour at al., Pharmacol. Biochem.Behav., 6:351-353 [1977]). Each mouse was placed alone on top of one offour wire mesh screens, each measuring 13×13 cm, that were mountedhorizontally on a metal rod 31 cm above the tabletop. The apparatus wasthen slowly rotated 180 degrees so that the mouse was suspended upsidedown on the bottom of the screen. The natural tendency is for the mouseto climb on top of the screen. After 60 seconds, each animal wasassigned to one of two groups: (1) animals that climbed to the top; and(2) animals that clung to the bottom or fell off the screen. An animalwas considered to have passed the test if it was able to climb back ontop of the screen with all four paws within 60 seconds. A cushion,placed beneath each screen to prevent injury to an animal should itfall, was at a sufficient height so that the mouse would not elect todrop. All mice were pretested 24 hours before treatment and only thosecapable of climbing to the top of the screen in the pretest were used inthis experiment.

FIG. 18 shows the effect of genistein on motor coordination as measuredby the inverted screen test for mice evaluated on days 1, 4, and 14after acute subcutaneous administration of saline, PEG vehicle, or 100,200, or 400 mg/kg of genistein. As indicated, there were no significantdifferences among groups.

FIG. 19 shows the effect of genistein on the motor coordination usingthe inverted screen test for mice evaluated on days 1, 4, and 14 afteracute oral administration of saline, PEG vehicle, or 100, 200, or 400mg/kg of genistein. As indicated, there were no significant differencesamong groups.

IV. Body Weight

The same non-irradiated animals used for the grip strength and invertedscreen tests were weighed throughout the 14-day period followinginjection. Clinical signs such as lethargy, fur condition, and generalwell-being were monitored at the time of weighing.

FIG. 20 shows the mean (SEM) body weight of mice administered an acutesubcutaneous dose of saline, PEG vehicle, or 100, 200, or 400 mg/kg ofgenistein. As indicated, there were no significant differences amonggroups.

FIG. 21 shows the mean SEM body weight of mice administered an acuteoral dose of saline, PEG vehicle, or 100, 200, or 400 mg/kg ofgenistein. As indicated, there were no significant differences amonggroups.

V. Testes Weights and Histopathology

On day 14 after injection, all mice from the two control groups (salineand PEG) and those from the high-dose group (400-mg/kg genistein) wereeuthanized and necropsied. Following the gross examination of eachanimal, tissues from the testes, liver, adrenal gland, mesenteric lymphnode, spleen, and bone marrow of the femur and sternum were collected,fixed in buffered formalin, paraffin embedded, sectioned, and stained byhematoxylin and eosin using methods known in the art. The wet weight ofboth testes without epididymes was determined before fixing in formalin.A board-certified veterinary pathologist examined all tissues. Theresults indicated that all gross necropsies and histopathology werenormal.

FIG. 22 shows the effect of a single oral gavage or subcutaneousinjection of saline, PEG vehicle, or 400 mg/kg of genistein on testesweights. Weights reflect the sum, 14 days after injection, of bothtestes with epididymes removed. Vertical lines represent the mean ″ SEM.As indicated, there were no significant differences among groups.

VI. Statistical Analysis

An analysis of variance and the Fisher's Least Significant Differencetest was used to statistically analyze grip strength, body weight, andtestes weight. The Fisher's exact test was used for analysis of theinverted screen test and 30-day survival data.

The results indicated that acute oral or subcutaneous doses (100-, 200-,400-mg/kg) of the soy isoflavone genistein administered tonon-irradiated adult male mice resulted in no adverse clinical signs orbehavioral toxicity. Acute oral or subcutaneous doses of 100-400 mg/kgof genistein resulted in no changes in body weight. There were noeffects from PEG vehicle or 400 mg/kg (high dose) of genistein on testesweights after an acute oral or subcutaneous administration of genisteinwhen compared with the testes weights for the saline control group. Thegross necropsy and pathological examination of adult mice treated with400 mg/kg of genistein or PEG vehicle revealed no abnormalities intissue morphology.

Example 7 Determination of Optimal Subcutaneous Protective Dose

In this Example, experiments conducted to determine the optimal dose andthe range of doses wherein a single subcutaneous dose of genisteinadministered 24 hours before radiation exposure protects mice againstradiation injury, the following experiment are described.Radioprotection was measured by 30-day survival. A dose-responseexperiment was performed where the radiation dose was 9.5 Gy 60-Cobaltand the dose rate was 0.6 Gy/min. The radiation procedure used was thesame as described above.

In these experiments, male CD2F1 mice (N=16-48/group) were administereda single subcutaneous dose of genistein in PEG-400 vehicle. Each mousereceived either saline, PEG-vehicle, 3.12, 6.25, 12.5, 25, 50, 100, 200or 400 mg/kg genistein administered subcutaneously 24 hr before 9.5 Gyat 0.6 Gy/min. The percent surviving for each of these group after 30days was: saline=8%, PEG-400 vehicle=15%, 3.125 mg/kg genistein=6%, 6.25mg/kg=0%, 12.5 mg/kg=19%, 25 mg/kg=60%, 50 mg/kg=56%, 100 mg/kg=65%, 200mg/kg=91%, and 400 mg/kg=85%, as shown in FIGS. 23 and 24. These datademonstrate that doses of 25 mg/kg genistein or higher are significantly(p<0.001) better than vehicle in protecting mice from radiation injury.

In summary, the present invention provides numerous advances andadvantages over the prior art, including methods and compositions forthe radioprotection. All publications and patents mentioned in the abovespecification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in diagnostics, cell culture, and/or related fields areintended to be within the scope of the following claims.

1. A method of enhancing the 30 day survival of an animal exposed to anacute, lethal dose of ionizing radiation comprising administering to theanimal a therapeutically effective amount of natural or syntheticgenistein, 6″-O-Mal genistein, 6″-O—Ac genistein, daidzein, 6″-O′ Maldaidzein, 6″-O—Ac daidzein, glycitein, glycitin, 6″-O-Mal glycitin,biochannin A, formononetin, or derivatives, analogs, or mixturesthereof.
 2. The method of claim 1 wherein the dose of ionizing radiationnormally produces lethality in said animal within a time period lessthan a year.
 3. The method of claim 1 wherein the administration is madeprior to the exposure.
 4. The method of claim 1 wherein theadministration is made 10 minutes to 96 hours subsequent to commencementof the exposure.
 5. The method of claim 1 wherein the administration ismade after the exposure.
 6. The method of claim 1 wherein theadministration is made from about 1 minute to 48 hours after radiationexposure.
 7. The method of claim 1, wherein said natural or syntheticgenistein, 6″-O-Mal genistein, 6″-O—Ac genistein, daidzein, 6″-O′ Maldaidzein, 6″-O—Ac daidzein, glycitein, glycitin, 6″-O-Mal glycitin,biochannin A, formononetin, or derivatives, analogs, or mixtures thereofis dissolved in a vehicle.
 8. The method of claim 1, wherein saidnatural or synthetic genistein, 6″-O-Mal genistein, 6″-O—Ac genistein,daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein, glycitein, glycitin,6″-O-Mal glycitin, biochannin A, formononetin, or derivatives, analogs,or mixtures thereof is part of a composition further comprising one ormore pharmaceutically acceptable carriers, excipients, auxiliaries, anddiluents.
 9. The method of claim 1, wherein said natural or syntheticgenistein, 6″-O-Mal genistein, 6″-O—Ac genistein, daidzein, 6″-O′ Maldaidzein, 6″-O—Ac daidzein, glycitein, glycitin, 6″-O-Mal glycitin,biochannin A, formononetin, or derivatives, analogs, or mixtures thereofis systemically administered.
 10. The method of claim 1, wherein saidnatural or synthetic genistein, 6″-O-Mal genistein, 6″-O—Ac genistein,daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein, glycitein, glycitin,6″-O-Mal glycitin, biochannin A, formononetin, or derivatives, analogs,or mixtures thereof is administered to said animal in a single dose. 11.The method of claim 1, wherein said natural or synthetic genistein,6″-O-Mal genistein, 6″-O—Ac genistein, daidzein, 6″-O′ Mal daidzein,6″-O—Ac daidzein, glycitein, glycitin, 6″-O-Mal glycitin, biochannin A,formononetin, or derivatives, analogs, or mixtures thereof isadministered to said animal in multiple doses.
 12. The method of claim1, wherein said natural or synthetic genistein, 6″-O-Mal genistein,6″-O—Ac genistein, daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein,glycitein, glycitin, 6″-O-Mal glycitin, biochannin A, formononetin, orderivatives, analogs, or mixtures thereof is contained in one or morepills, capsules, liquids, gels, powders, suppositories, suspensions,creams, jellies, aerosol sprays, or dietary supplements.
 13. The methodof claim 12, wherein said dietary supplement comprises an unprocessedsoy food.
 14. The method of claim 12, wherein said dietary supplementcomprises isolated soy protein.
 15. The method of claim 1, wherein saidnatural or synthetic genistein, 6″-O-Mal genistein, 6″-O—Ac genistein,daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein, glycitein, glycitin,6″-O-Mal glycitin, biochannin A, formononetin, or derivatives, analogs,or mixtures thereof is utilized in amount of from about 0.1 mg to about2000 mg.
 16. The method of claim 1, wherein said the dosage of saidnatural or synthetic genistein, 6″-O-Mal genistein, 6″-O—Ac genistein,daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein, glycitein, glycitin,6″-O-Mal glycitin, biochannin A, formononetin, or derivatives, analogs,or mixtures thereof administered to said animal comprises from about 30mg/day to about 200 mg/day of said isoflavonoid.
 17. The method of claim1, wherein said natural or synthetic genistein, 6″-O-Mal genistein,6″-O—Ac genistein, daidzein, 6″-O′ Mal daidzein, 6″-O—Ac daidzein,glycitein, glycitin, 6″-O-Mal glycitin, biochannin A, formononetin, orderivatives, analogs, or mixtures thereof is administered to said animalis in a dosage of an effective amount less than about 400 mg/kg/day ofthe body weight of said animal.
 18. The method of claim 1, wherein saidadministering is subcutaneous injection, oral administration,intravenous administration, rectal administration, vaginaladministration, topical administration, intramuscular administration,intranasal administration, transdermal administration, subconjunctivaladministration, intraocular administration, periocular administration,retrobulbar administration, subretinal, suprachoroidal administration,or intrathecal administration.
 19. The method of claim 1, wherein saidadministering is administration from mechanical reservoirs, devices,implants, or patches.
 20. The method of claim 1, wherein said radiationis ionizing radiation, alpha radiation, beta radiation, gamma radiation,or neutrons.